Microfluidic device and methods for construction and application

ABSTRACT

A microfluidic device comprises first and second inlet passages ( 13 ) for respective immiscible fluids, these inlet passages merging into a third passage ( 8 ) along which the two fluids flow under parallel laminar flow conditions, the third passage being formed with a constriction or other discontinuity ( 9 ) causing the two fluids to form into a flow of alternate segments.

FIELD OF INVENTION

The present invention relates to microfluidic devices and assemblies,methods for their construction with preferred embodiments andmethodologies for the functional operation of such.

BACKGROUND

The miniaturisation of chemical processes onto chip-based platformsenables a plethora of novel industrial applications of existing and newchemistry, biochemistry, biomolecular science and particle science inboth analysis, synthesis, assembly, decision making and computing. Forexample, molecular synthesis in micron-scale reactors benefits from (i)fast reaction kinetics, and (ii) high specific-areas which facilitatesgreater control of and/or the use of highly exothermic reactions. Manyadvances in so-called, microfluidics have been made in recent years.Nevertheless, known devices and methods for the manipulation of fluidsin miniaturised tubes, ducts and vessels have not met all therequirements of industry. For example, sterilisation of, and themaintenance of an inert atmosphere in, microfluidic ducts remains aproblem hampered by the materials from which many devices have beenconstructed. Additionally, the use of highly corrosive fluids and hightemperatures again requires very strict attention to the materials fromwhich devices are fabricated, which, in turn, has serious implicationfor mass-manufacture of such devices and assemblies of such.Accordingly, special attention must be made to the suitability ofconstructional materials, the unit costs of manufacture, the rapidity ofmanufacture, including tooling time for mass-production, and thetranslation of prototyping methods to mass-production. Constructionalmaterials may include a glass, ceramics, stainless steel and othermetals or alloys, silicon, polymers, paper and others. Glass-basedsubstrates have been successfully manufactured, but limited, to someextent, by the complexity of 3-dimensional geometry that iscost-effectively feasible. Also, external fluidic interconnect solutionsremain crude. Photostructurable glasses (e.g. Fotoran made by Schoot)are very expensive, sometimes 200× the cost of polymer substrates, andrequire several expensive and hazardous processing steps (e.g. use ofHF) and specialised equipment (e.g. quartz based optics forlithography). Stainless steel chips can be manufactured but are limitedwith respect to 3-dimensional geometries attainable and the surfacequality possible, even with MicroElectroDischarge Machining, isfrequently of insufficient resolution for microfluidic applications. Thetechnique suffers from high unit-cost production and very limitedavailability of high resolution machining tools. Silicon basedmicrofluidic devices, such as microreactors, have been made and benefitfrom available tools for silicon micromachining and fusion/anodicbonding procedures for bonding together multilayered devices. However,silicon is relatively expensive for mass fabrication of relativelylarge-format chips which may sometimes have a short-lifespan. Inaddition, with exceptions, interconnect solutions remain inelegant andlow-pressure and silicon denies the use of high field strength electricfields for the generation of electro-kinetic flow and certain molecularpurification processes.

Many polymers (e.g. polysulphone, polycarbonate, polymethylmethacrylate)have been utilized for the fabrication of microreactors but most havebeen unsuitable for use with very aggressive liquids such as acids (e.g.nitric acid) and solvents (e.g. acetonitrile). In addition, the presenceof certain substances incorporated into the polymer matrix, such asplasticisers, may cause contamination during usage, as those compoundsleach from the substrate matrix into the fluids within the ducts on thechip. Particularly, for many synthetic reactions the preferred substratematerial would be a fluoropolymer such as polytetrafluoroethylene (C₂F₄)_(n) [PTFE]. However PTFE and related variants are less easilymicromachined to provide fluidic ducts of micron sized dimensions andvery difficult to join with itself to form enclosed microreactor ducts.It is a purpose of the current invention disclosed herein to provide acost-effective resolution to the latter technical problems and enablethe manufacture of suitable chip-based platforms for a wide range ofindustrial-scale diagnostic and synthesis operations.

Additionally, fluid flow in microscale ducts is characterised by laminarflow conditions resulting from characteristically low Reynolds numberregimes. This causes a problem with mixing of fluids and it is a purposeof the invention disclosed herein to provide a solution to thattechnically limiting issue. Also, fluid flow in micron scale ducts isusually characterised by continuous streams of a given fluid phase. Acontrasting method is where immiscible phase fluids are caused to flowalong a duct in serial discontinuous aliquots. The generation of suchsegmented flow streams can be enabled by bringing together two streamsof immiscible fluid and causing them to merge at a so-called T-junction.This methodology has not met all the needs of industry. For example,such device configurations are frequently only stable for a narrow rangeof absolute flow rate conditions and relative flow rates of theimmiscible phase liquids. In particular, it can be difficult to controlthe generation of segmented flow streams with equal volumes of theimmiscible phases, especially at low flow rates required by manyapplications. In addition, back-pressure can be considerable, especiallyin ducts of narrow (<100 microns width, depth, both or diameter) andvery narrow (<25 micron width, depth, both or diameter) dimensions. Itis, therefore, a purpose of the invention disclosed herein, to provideimproved solutions for the generation and subsequent manipulation ofsegmented flow streams in micron scale ducts.

Devices for the manipulation of fluids may be used for analytical andsynthesis purposes. Frequently, for a wide range of functionaloperations in both analytical and synthesis techniques it is necessaryto elute precise volumes of fluids in a highly repeatable manner. Forexample, in titrations, ‘split and mix’ procedures, formation ofmicroparticles such as artificial cells and nanoparticles such asquantum dots. Because the volumes of liquids are usually very small itis frequently difficult to meet the exacting requirements of industryand solutions to date are generally insufficient to meet all needs. Itis a further purpose of the invention disclosed herein to provide deviceconfigurations and methods to improve substantially on those currentlyavailable. Furthermore, notwithstanding that devices and methodscurrently available for the controlled volumemetric elution of liquids,do not meet current needs, the subsequent manipulation of small liquidvolumes also requires improvements. In particular, there is a need toimprove on techniques for altering the morphology of liquid samples,their conversion to non-liquid forms and the ability to encapsulate suchsmall samples with films of other materials. It is also a furtherpurpose of the invention disclosed herein to provide further devices andassociated methods to meet these needs.

SUMMARY OF THE INVENTION

In accordance with a first aspect, a fluid manipulation device isdescribed, which comprises a device composed of, at least, two distinctfluoropolymer based substrate layers which may be composed of bulkfluoropolymer or fluoropolymer- or fluoropolymer-based coatings appliedto other non-fluoropolymer bulk material substrate layers. Thefluoropolymer bulk material layer(s) or applied coatings may includePTFE, Teflon®, Teflon®AF, Teflon®NXT, Teflon® G, Teflon® PFA, Teflon®PFA HP Plus, Dyneon™, CYTOP®, Teflon® PFTE-silicone adhesive film(product 5490 of 3M). Additionally, in accordance with this firstaspect, the fluid manipulation device is provided with at least twoducts, for the passage and/or storage of immiscible fluids.

It will be understood by those skilled in the art that either of theimmiscible fluids may include aqueous matrices, supercritical fluids,supercritical Helium-3, organic solvents, ionic liquids, inert fluidssuch as perfluorinated alkanes, perfluoroethers (including but notlimited to Galden®, Fluorinert™), oils, acids, gasses, suspensions ofliving (including cryo-preserved) cells, organelles and tissues,suspensions of preformed particulate materials such as chromatographicseparations media, tissue scaffold precursors, etc.

It will further be understood by those skilled in the art that in turnthe liquids may incorporate other components including (but not limitedto): smaller particles of polymers (gelled or crosslinked) includingnon-biodegradable polymers such as (but not exclusively) ethylene vinylacetate, poly(meth)acrylic acid, polyamides; biodegradable polymersincluding synthetic polymers such as polymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters; natural polymers suchas alginate and other polysaccharides including dextran and cellulose,collagen, chemical derivatives thereof, albumin and other hydrophilicproteins, zein and other prolamines and hydrophobic proteins, copolymersand mixtures thereof; bioadhesive polymers including bioerodiblehydrogels, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates); antibodies, enzymes, abzymes,liposomes, antibiotics, nucleic acids and associated promoters,supercoiled DNA, oligonucleotides with <30 nucleotides, genes of morethan 30 nucleotides with and without expression promoters; a wide rangeof pharmaceutical molecules, living cells, organelles, bacteria,viruses, viral vectors, metallic particles, magnetic particles, quantumdots, radioactive particles or radioactive-tagged molecules,fluorescent- chemi-luminescent- and/or bio-luminescent particles,molecularly imprinted polymers, tissue scaffold precursors,semiconductor devices, micron-scale RF transponders, liquid crystals,porous silica particles such as beads, porous silicon particles andothers.

The two ducts are arranged to be separated by a thin partition(typically less than 500 um, preferably less than 10 um and mostpreferably less than 1 micron) until a point at which the partition isno longer present. The two parallel, closely associated ducts join to athird duct (hereafter called the commmon duct) measuring, optionally 500microns to 100 nanometeres in either cross-sectional dimensions ofwidth, depth, mean diameter, but of a length, at least, ten times theminimum width of either the two preceding ducts, so that the differentfluids moving in parallel flow formation along the third duct attain avery stable flow profile free from shear forces and microturbulence attheir interfacial juncture. The cross-sectional area of the third ductis preferably, equal to the sum of the cross-sectional area of the twopreceding ducts. The third duct axially joins a fourth duct (hereaftercalled the segmented flow duct) that is of cross-sectional area andwidth at least 25% most preferably 50%, that of the third duct. At thejuncture of the said common duct and constriction duct, the permanent ortemporary, 2- or 3-dimensional, constrictions in cross-sectionalgeometry are configured such that when two or more streams of immisciblefluids are caused to move within the common duct the fluids may beforced from a laminar unmixed flow condition to form contiguous serialsegments of the immmiscible liquids in the fourth duct. The immiscibleliquids may be caused to move within the ducts by means of appliedpressure- (negative or positive), electrokinetic forces (where chargedfunctional groups are provided on the walls of the fluid bearing ducts),displacement (e.g. by the action of a plunger caused to move by theapplication of a mechanical force from a human being) or centrifugalforces (e.g. by means of rotation about an axis). The absolute rate offlow of fluid segments may be adjusted by varying, in equal amounts, theflow rates of the immiscible fluids. The relative volumes of theimmiscible liquids formed as fluid segments may be adjusted by varyingthe relative flow rates of the same immiscible liquids. At the point atwhich the two or more ducts merge the immiscible fluids remainsubstantially in parallel (laminar) formation and continue in suchformation as they are caused to move along the common duct. When orwhere the common duct(s) is/are physically constricted (permanent ortemporarily) the liquid components are forced together in a moreconstrained environment. At the point(s) of constriction the laminarflow may become interrupted such that one liquid will move into thenarrower section of the duct first to be followed by aliquots of thesecond and other immiscible liquids in a manner determined by therelative contact angles of the fluid component phases, the surfaceenergy of the fluoropolymer material, the force applied to propel thefluids, elastic properties of the component liquid phases and theprecise 2- and 3-dimensional geometrical nature of the physicalconstriction. This allows the selective passage of given phase liquidsto pass along the duct establishing a repeated and ordered segmentedstream of liquid phase materials. Because the flow is manipulated in acontrolled volume environment and because all the environmentalparameters (e.g. but not limited to pressure, flow-rate, temperature,humidity, surface energy) can be finely controlled, fluid segments areproduced to a highly accurate volume and morphological specification.This fine control of fluid segment volume and geometrical morphologygives rise to many advantages that can be of benefit to analytical andsynthesis operations when conducted in the fluid based device. Theabsolute lengths of fluid segments may be controlled in a time-varyingdynamic manner by controlling the absolute flow rate of the fluids intoand along the common fluid duct. The relative lengths of the fluidsegments may be determined by providing ducts of differingcross-sectional area for the separated flow streams prior to merger inthe common fluid duct. The relative lengths of the fluid segments may bedetermined in a time-varying dynamic manner by controlling the relativeflow rates of the separated flow streams prior to merger in the commonfluid duct.

Additionally, in accordance with this first aspect, the saidconstriction feature is utilised, at least (but not exclusively), forthe controlled volume elution of immiscible fluid phase components.Additionally, in accordance with this first aspect, the device isdesigned and manufactured, specifically and uniquely, for the creationof, and functional operation with, at least one segmented flow stream ofimmiscible fluid-based matrices, which are favourably enabled by the useof low surface energy fluoropolymer-based manufacturing materials.Additionally, in accordance with this first aspect, the said fluidicducts are machined, most preferably by means of reactive ion etching,even more preferably with the assistance of an inductively coupledplasma, even more preferably where the fluoropolymer-based or coatedsubstrates and/or reactive plasma are cooled with a cryo-facility. Aparticular advantage of utilising reactive ion etching of PTFE andrelated polymers is that it is frequently difficult to micromachine byother means.

For instance, because pure PTFE it is not a thermoplastic it is noteasily moldable and as such does not lend itself to microstructuring byembossing or injection molding techniques. When ablated by lasermachining [e.g. copper vapour, excimer] the ablated surface is oftenhighly irregular and generally unsuited to microfluidics chemistry andother applications. Furthermore, reactive ion etching is a parallelprocess in that all areas of substrate exposed to the plasma are etchedat the same time which renders it suitable to manufacturing ofsubstrates suitable for microfluidics, microchemistry and otherapplications where a relatively large substrate format is required. Thiscontrasts with excimer laser micromachining which is frequently used(though not always) in a serial manner where the laser beam is movedover a workpiece to define a geometry.

Additionally, the reactive ion etching process lends itself tomass-production by virtue of available equipment commonly employed forsilicon and silica micromachining. Automated cassette-loading systemswith large wafers could facilitate high-volume production with theadvantage that complex, high aspect ratio, nanoscale features could beincorporated which is not easily achieved with medium volumemicromanufacturing techniques such as injection micromolding. Reactiveion etching of fluoropolymers may be used as both a prototyping andmass-manufacturing process. This provides for a seamless transfer ofprocessing technology to different scales of production. This holds anadvantage over embossing and injection molding where separate tools havefirst to be constructed which is both time-consuming and expensive.Because manufacturing costs of components made by etching aredetermined, in part, by processing time, accelerated etching timesallows faster processing operation to be done with the result of higherunit throughput. Unit costs of manufacture are thus reduced. Lesspreferably, the ducts may be machined by CNC milling, preferably wheresubstrates are cooled to lessen the surface roughness of the finishedsurfaces. Less preferably the said ducts may be machined inthermoplastic variants of fluoropolymer based substrates by means of hotembossing, injection molding, laser ablation (excimer or copper vapour)and broad area ion-beam milling.

Additionally, in accordance with this first aspect, the immisciblefluids are located and manipulated within the said ducts, the devicehaving no operative fluid ports for communication external to thedevice. Additionally, in accordance with this first aspect, the deviceincorporates a passive (batteryless and wireless) radio frequencytransponder device to provide individual device identification.Additionally, in accordance with this first aspect, thefluoropolymer-based or fluoropolymer-coated layers are encased undercompression within a recess machined in between two or more encasementlayers of a dissimilar thermoplastic (e.g. PMMA, polycarbonate) that maybe mechanically bonded together or joined by thermal, thermo-compressionor thermally assisted (e.g. by inductive heating) compression bonding.The fluoropolymer and/or fluoropolymer based layers are provided with a(combined) slightly larger (by 10 microns and up to 100 microns)thickness dimension than the depth of a recess provided in the separatecasement. During thermal bonding of the casement the fluoropolymer-basedinsert becomes embedded within it such that upon cooling thefluoropolymer-based layers are sandwiched at high pressure. This resultsin a mating between the fluoropolymer-based surfaces that are fluidtight at levels highly useable for low-pressure applications,particularly in chemistry and biotechnology. Preferred embodiments ofthe device may be utilised for a wide range of industrial research,development and manufacturing operations, as a singular device, or as 2or more operating in serial or parallel arrangements and/or as a deviceinterfaced with other equipment. Additionally, in accordance with thisfirst aspect the device is manufactured substantially in a planarlayer-like format, each of which are of a geometrical area >350 squaremillimetres, typically 8000 square millimetres and 32000 squaremillimetres, most preferably in circular format, less preferably inrectangular format, each layer being at least 40 microns in thickness,with one layer being at least 475 microns in thickness.

In accordance with another aspect, the fluid manipulation device isconfigured with a port suitable for fluidic communication with theexternal environment and/or equipment, and/or mammalian body. Suchequipment may include, but not be limited to, rotary, piston and otherdisplacement pumps; vacuum lines, gravitationally driven fluid streams;analytical apparatus for mass spectrometry, liquid chromatography,particle characterisation, nuclear magnetic resonance, impedancespectroscopy, UV/VIS/IR absorption; and sample storage (including butnot limited to microtitre plates, vials, collection vessels, porousmaterials in bead, strip, card and disc-like formats, dry storage platesincluding porous silicon microspot arrays).

In accordance with another aspect, the fluid manipulation device may beconfigured with two ports suitable for said fluidic connection toexternal environment, mammalian body and equipment.

In accordance with another aspect, the fluid manipulation device may beconfigured with three ports suitable for said fluidic connection toexternal environment, mammalian body and equipment.

In accordance with another aspect, the fluid manipulation device may beconfigured with more than three ports suitable for said fluidicconnection to external environment, mammalian body and equipment.

In accordance with another aspect, the fluid manipulation device may bemanufactured, such that one or more substrate layers, formed of bulkfluoropolymer or fluoropolymer coated material, utilises a thermoplasticfluoropolymer that melts when raised above its glass transitiontemperature. The juxtaposed layers may be hermetically joined by theinductive heating (upon exposure to strong electromagnetic fields) of ametallic (e.g., but not limited to, titanium, aluminium, chromium)interlayer film deposited on to one or more of the said layers, andwhich, upon inductive heating to above the glass transition temperature,causes local melting of the thermoplastic fluoropolymer materials, whichupon subsequent cooling (after removal of the inductive heatingelement), forms a hermetic and mechanically secure seal.

In accordance with another aspect, the fluid manipulation device may bemanufactured, such that one or more substrate layers, formed of bulkfluoropolymer or fluoropolymer coated material, utilises a thermoplasticvariant of fluoropolymer that melts when raised above its glasstransition temperature. The juxtaposed layers may be hermetically joinedby the combined application of heat and pressure, raising the surfacetemperatures of the layers where they adjoin to at least 5° C. above theglass transition temperature. This causes local melting of thethermoplastic fluoropolymer materials, which, upon subsequent cooling,forms a hermetic and mechanically secure seal.

In accordance with another aspect, the fluid manipulation device may beprovided with the additional incorporation of protrusions of thethermoplastic encasement material, that, when assembled, fit through viaholes provided in the fluoropolymer-based or -coated substrates, andmate with corresponding protrusions from the opposite side. This matingmay be caused by simple butt joints or by a number of geometricalarrangements where ‘plug and socket’ type modifications to theprotrusions are made. These protrusions may be securely butnon-permanently joined, simply by mechanical means, and/or madepermanent by thermo-compression bonding. The joined protrusions act toprovide additional strength to the hybrid assembly particularly on largeformat structures and prevent fluid escape between the layeredfluoropolymer-based or -coated parts. Small through-vias may be providedthrough ‘plug and socket’ type protrusions such that an oversized pin(preferably made, though not exclusively, from stainless steel) driventhrough the vias will enhance physical contact and the security of thefastening achieved, or by use of a bolt and nut tightened to secure amechanical and hermetic fastening.

In accordance with another aspect, the fluid manipulation device may bemanufactured where one or all of the fluoropolymer based layerscomprises a fluoropolymer-based thin (typically 1-3 microns) film (e.g.Teflon® AF) film applied to an optically transmissive material (e.g. butnot limited to borosilicate glass, Pyrex, QC PMMA) such that the fluidcontained within the fluid ducts and receptacles is enclosed completelyby fluoropolymer based materials whilst allowing for the passage oflight which may be used for imaging, and/or photo-excitation of devices(e.g. actuators) or phenomena (e.g. photocatalysis, gelling). This thinfilm may be applied by means of various techniques such as spin-coating[solutions or nanoparticle suspensions], RF magnetron sputtering orPlasma Enhanced Chemical Vapour Deposition, optionally followed by anannealing process, also optionally followed by exposure to an oxygen oroxygen/ammonia plasma to enhance adhesive and/or (chemical)functionalisation qualities.

In accordance with another aspect, the fluid manipulation device may beprovided with fluidic ducts and receptacles that are machined in bothsides of the fluoropolymer based or coated substrate layers providingfor double sided fluidic substrates. In such devices a thirdfluoropolymer-based or -coated layer is required to provide a fluidicenvironment where all parts of the fluidic system are provided byfluoropolymer-based materials.

In accordance with another aspect, the fluid manipulation device may beprovided with one or more fluidic vias through the substrate layersproviding fluidic communication between both sides of the substrate.

In accordance with another aspect, the fluid manipulation device may bemanufactured with two or more single- or double-sided fluidic substratelayers that may be stacked upon each other, directly or indirectly(optionally, with a separating layer) to create a multi-layer assemblage(e.g. up to 1000 or more substrate layers). These layers may haveidentical or dissimilar function. Such devices may incorporate one ormore additional substrate layers, the function of which is to act as amanifold for distributing fluid based matrices from one or more inputand output tubes to one or many ducts configured to generate segmentedflow streams.

In accordance with another aspect, the fluid manipulation device may bemanufactured such that, additional, smaller modules may be attachedaboard the device so as to provide additional functionality. Thesemodules may incorporate sensors (e.g. impedance sensor measuring liquidpurity), actuators (e.g. diverters of fluid flow) reservoirs of reagents(e.g. fluids, particles, etc.) or may function to provide clusters ofone or more additional input-output fluid ports to the fluidic substratelayers or stack of substrate layers. These modules may be attached bymeans of one or more plug-like projections from the smaller moduleswhich respectively mate with socket-like receptacles provided in thesubstrate layers, or encasement layers of the device. The modules may bestacked one upon another as described in International Patentapplication WO02/060810. In a preferred embodiment, 6 plug-likeprojections are arranged peripherally and circumferentially on theunderside of disc-like modules at separation angles of 60°, but thenumber, position and format may take many variant forms. The plug-socketconnectors allow for mechanical fastening, fluid transport, electricalconnectivity, optical connectivity, the intimate application ofelectrical and magnetic fields and the intimate application of inductiveheating.

In accordance with another aspect, the fluid manipulation device may bemanufactured where the encasement layers are manufactured from anon-polymer material (most preferably, though not exclusively stainlesssteel) joined not by thermocompression bonding but rather through theuse of one or more bolts extending between casement layers so as to forma mechanically tight assemblage of fluid substrate layers. In accordancewith another aspect, the fluid manipulation device may be manufacturedwhere no encasement layer is provided, the fluid substrate layers beingpreferably constructed from thermoplastic fluoropolymer (e.g. Teflon®AF, Teflon® NXT, Teflon®G, Teflon® HP, Teflon® HP Plus) bulk materialsor thermoplastic fluoropolymer coated material of another polymer (e.g.,but not exclusively, polycarbonate, polymethylmethacrylate), the saidlayers being joined by thermal, thermally assisted, ultrasonicallyassisted or thermocompression bonding.

In accordance with another aspect, the fluid manipulation device may beprovided with a fluid port such that one of the fluid phase componentsis introduced on to the device from an external supply.

In accordance with another aspect, the fluid manipulation device may beprovided with two fluid ports such that two of the fluid phasecomponents are introduced on to the device from external supplies.

In accordance with another aspect, the fluid manipulation device may beprovided with sufficient ports that all of the fluid phase componentsare introduced on to the device from external supplies.

In accordance with another aspect, the fluid manipulation device may beprovided with a fluid port such that one of the fluid phase components,or products of, may be exported from the device to an external location(such as a collection vessel, instrument, mammalian body, atmosphere, orvacuum).

In accordance with another aspect, the fluid manipulation device may beprovided with two fluid ports such that two of the fluid phasecomponents, or products of, may be exported from the device to anexternal location (such as a collection vessel, instrument, mammalianbody, atmosphere, vacuum).

In accordance with another aspect, the fluid manipulation device may beprovided with many fluid ports such that three or more of the fluidphase components, or products of, may be exported from the device to anexternal location (such as a collection vessel, instrument, mammalianbody, atmosphere, vacuum).

In accordance with another aspect, the fluid manipulation device may bemanufactured such that one or more geometrical constrictions in a fluidduct, designed to cause segmented flow of immiscible fluids, arereplaced or enhanced with a permanent step change in surface energyachieved by the differential patterning of the surface energy states.These altered energy states may be achieved in a time-non-varying mannerby spatially-variable exposure of the surfaces to reactive plasmas, thegrafting of functional moieties or the selective patterning of adissimilar fluoropolymer film over the substrate layer. One example iswhere a thin film of Teflon® AF (a copolymer of Teflon® andperfluoro-2,2-dimethyl-1,3-dioxole) is used to coat the surface of thefluidic ducts but where an electronegative charge is selectivelyimplanted, in a spatially-variable manner, in the film by, for example,use of a back-lighted thyratron electron gun.

In accordance with another aspect, the fluid manipulation device may bemanufactured such that one or more geometrical constrictions in a fluidduct, designed to cause segmented flow of immiscible fluids, is replacedwith a time-varying step change in surface energy that may be actuatedto cause otherwise laminar flows of immiscible phase liquids toreconfigure to segmented flow. This may be achieved by electro-wettingwhere one or more insulated electrodes apply and determines the shape ofan electric field at given points within the fluidic duct(s) so as tocause, otherwise, laminar flows of immiscible phase liquids toreconfigure to segmented flow. A similar result may also be achieved byphoto-wetting where one or more addressable photo-responsive surfacesare incorporated within the common fluidic duct such that when excitedby illumination with incident light, the altered surface energy states,causes otherwise laminar flows of immiscible phase liquids toreconfigure to segmented flow. These altered energy states may beinduced in, for example, spatially-variably deposited thin films ofsilicon (e.g. by electron beam deposition) or photo-responsivefunctional moieties grafted on to the surface of the fluoropolymer orfluoropolymer coated ducts.

In accordance with another aspect, the fluid manipulation device may bemanufactured where the geometrical constriction in a fluid duct,designed to cause segmented flow of immiscible fluids, is enhanced orreplaced with a cruciform junction of fluidic ducts whereby a separateduct crosses the path of the common fluidic duct. An intermittent DCvoltage applied to the separate fluid duct which crosses the commonfluid duct will cause the momentary, preferential, electro-kineticmovement of aqueous fluids from the common fluidic duct, resulting inthe conversion of otherwise laminar flows of immiscible phase liquids toreconfigure to segmented flow within the common fluidic duct.

In accordance with another aspect, the fluid manipulation device may beprovided with one or more ducts, in which segmented flow has beencreated, which said ducts split with partitions at a given point, toprovide two or more separate ducts (referred to here for convenience as‘shredding ducts’) which are smaller in cross-sectional dimensions. Thispartitioning into ‘shredding’ ducts cause fluid segments to break intosmaller segments thus having a ‘shredding effect’. One or more of thesmaller ducts may also be similarly partitioned again, so causingfurther division of the fluid segments. The 2- and 3-dimensionalgeometries of the ‘shredding ducts’ may be selected such that therelative morphology of the fluid segments remains constant, or such thatthe morphology is significantly altered (for example to producefilamentous fluid segments). The smaller ducts may reduce in size to aslittle as 70 nanometres in width and 200 nm in depth.

In accordance with another aspect, the fluid manipulation device may beprovided with two or more ducts, each said duct carrying immisciblecomponent fluids, each of which have been configured to flow insegmented formation (hereafter called feeder ducts). The segmented flowducts merge to form a common duct resulting in the coalescence ofindividual fluid segments, of the same phase, from the two or morepreviously separated segmented flow (feeder) streams. Each coalescedfluid segment assumes the internal circulation pattern typical ofsegmented flow, thus causing rapid mixing within a given segment. Thedevice may be used to react/mix or dilute fluid phase components derivedfrom the feeder ducts. The device may be used to advantageously(rapidly) mix aqueous solutions/suspensions which, otherwise, wouldremain relatively unmixed for a long duration whilst constrained underthe laminar flow conditions that usually dominate in microfluidicenvironments. The device may be advantageously utilized to dilute onegiven fluid phase fed from one of the feeder ducts by altering thevolume ratio of the immiscible phase components in the other feederducts.

In accordance with another aspect, the fluid manipulation device may beprovided where the unit of operation for mixing dilution is replicatedat least two or more times on the same device substrate or stack ofsubstrates. Particularly, the multisite mixing and/or dilutioncapability allows the device to simultaneously mix and/or dilutereagents prior to dispensing into or onto a number of integral orexternal multiwell or multisite devices for the purposes of furtherprocessing operations (e.g. into the wells of a microtitre plate) orstorage (e.g. on to selective sites of a MALDI plate device, or a porousmaterial such as a bead, ceramic plate or layer of porous silicon).

In accordance with another aspect, the fluid manipulation device may beconfigured such that two or more aqueous flow streams are caused to flowalong a common duct, which merges (as a constriction- or T-junction)with one or more separate ducts carrying an immiscible fluid, resultingin the formation of a segmented flow stream, the internal vortex flowpatterns of which fluid segments causes the rapid mixing of the 2 ormore aqueous liquid matrices, which would, under laminar flowconditions, otherwise, remain unmixed for a considerable perioddepending upon the Reynolds Number.

In accordance with another aspect, the fluid manipulation device may bemanufactured such that a first duct carrying a segmented flow stream isjoined at a juncture, by one or more additional ducts, preferably inserial succession, at which points of juncture the first duct is,optionally, geometrically enlarged in cross-sectional area (either inwidth, depth or both), and where, during the passage of fluid flow alongthe first duct, supplementary aliquots of fluid from the additionalducts may be injected or added to the volume of selected, or all, fluidsegments in the first duct. The supplementary fluid added to the fluidsegments is caused to mix rapidly by virtue of the internal flow vortexwithin the component fluid segments. The flow of fluid from theadditional duct into the first duct may be caused by passive means wherelike solutions momentarily contact and ‘pull off’ an additional aliquotof fluid from the additional duct. Equally, the flow of fluid from theadditional ducts may be caused by propulsive methods including positivedisplacement pump, electrokinetic injection (e.g. actuated by monitoringthe passage of individual fluid segments in the first duct),piezoelectric actuator, and others.

In accordance with another aspect, the fluid manipulation device may bemanufactured with one or more pressure sensors in the form of capillarygas-filled manometers. These are constructed as blind tributary ductsarranged at any angle between 0° and 180° to the first microfluidic ductalong which fluids are caused to move. The first microfluidic duct maybe any duct formed on the device for manipulation of fluids where thereis non-segmented flow. The tributary ducts are preferably filled with arelatively inert gas such as, though not exclusively, argon, helium ornitrogen. Fluid within the first fluidic duct will rise into thetributary duct according to the prevailing pressure conditions. Thetributary ducts function according to the principle that a change in gasvolume provides a change in fluid pressure, i.e., an increase in fluidicpressure gives a decrease in gas volume. Therefore, by knowing the crosssectional area and length of the blind tributary ducts, the initialvolume can be calculated and with the displacement of the fluid in thechannel the increase/decrease of the pressure affected volume can becalculated. With liquid in the first microfluidic duct, the liquid levelwithin the tributary duct rises to an initial pressure setting.According to pressure changes (for example, during fluid movement causedby the application of pressure) the pressure of the fluid increases andthe fluid moves further along the tributary duct changing the volume ofthe trapped gas. Other geometrical arrangements of more than onetributary duct can provide information on fluid flow rate by indicatingthe pressure drop over predetermined length or over a fluid resistorcaused by a constriction in the first fluid duct. The capillarymanometers may be provide in a linear, serpentined, curved or coiledformat and meniscus movements visualised by an optical device such as aCCD.

In accordance with another aspect, the fluid manipulation device may beprovided with one or more peltier based microcooler devices which causeparts of the fluid duct(s) and their contents to be reduced intemperature. This may be particularly useful for a range of(bio)physiochemical operations such as replication of nucleotidesequences, protein crystallisation, polymer cross-linking by light- andion-exchange means, exothermic chemical reactions and others.

In accordance with another aspect, the fluid manipulation device may bemanufactured where a first duct carrying a segmented flow stream isconstricted and reduced in cross-sectional area (width, depth, both, ordiameter) so as to cause fluid segments formed in the first duct beforethe constriction, to change shape, such as becoming elongated or alteredin cross-sectional profile (for instance, assuming a stellar-like orcruciform section). Optionally, such fluid segments may be caused tobecome less fluid in nature by partial or total polymerisation and/orgelling, by the selective exposure to light (most preferablyultraviolet, less preferably visible and infrared light) temperaturealteration, ionic composition of external environment, or the presenceof gelling/polymerisation or encapsulation agents injected into the flowstream or directly into individual fluid segments.

In accordance with another aspect, the fluid manipulation device may bemanufactured where a first duct carrying a segmented flow stream isexpanded in geometrical cross-sectional area (either in width, depth,both or mean diameter) by an amount sufficient to cause fluid segmentsin the first duct, before the point of expansion, to change shape fromnon-spherical forms, to a spherical format, the boundary layers of eachsegment no longer assuming intimate shear contact with the duct walls.Configured in this manner, the rapid internal vortex flow that can beinduced within the fluid packets of segmented flow (due to shear contactwith the duct walls during passage along the said duct) is lost as fluidsegments assume a spherical format with no intimate shear contact withthe duct walls. Following this methodology contiguous fluid segmentsbecome converted to trains of spheres of highly similar volume. Thetrains of spheres may be collected for usage or further processed.

It will be understood by those skilled in the art, given the benefit ofthis disclosure, that an ability to produce droplets of highlyreproducible volume and shape has innumerable applications and useswhere size and shape affect their function. An example is the size ofparticles (e.g. liposomes, alginate beads) designed to deliverpharmaceuticals and genes to target tissues within the mammalian body.Similarly the production of droplets of highly reproducible volume andshape is particularly useful in the production of foodstuffs, cosmetics,detergents and skin-care products.

In accordance with another aspect, the fluid manipulation device may beused where the contents (or part) of liquid segments are polymerised orcrosslinked by exposure to electromagnetic radiation, [in the wavelengthrange 200-700 nm, most preferably in the long wavelength ultraviolet(LWUV) range or visible range, 320 nm or higher, and most preferablybetween about 365 and 514 nm]. Dyes and co-catalysts, such as amines,for initiating polymerisation may also be included in the liquidsegments such that upon exposure to visible or long-wave ultravioletlight, active species can be generated. Light absorption by the dyecauses the dye to assume a triplet state, which subsequently reacts withthe amine to form an active species which initiates polymerization.

In accordance with another aspect, the fluid manipulation device may beused where the contents (or part) of liquid segments are ionicallycross-linked by the exposure to appropriate gelling agents deliveredthrough a subsidiary duct, joining the main duct carrying the segmentedflow stream(s), at, or just after, the point of duct expansion. Oneexample to illustrate this is the gelling of (typically, 1-12%) alginatesolutions [which may in turn incorporate smaller particles or othercomponents as described herein], where a segmented flow stream of sodiumalginate and an immiscible phase such as an oil or perfluoroethers suchas Galdens®, Fluoinert™, or organic solvent such as methylene chlorideis created in a duct which incorporates an expansion zone. At theexpansion zone the segments of alginate emerge as droplets (preferably,though not exclusively as spherical droplets) suspended within the flowof the immiscible phase, and due to the added exposure to weak calciumlevels (typically 10 mM-200 mM Ca Cl₂) delivered in to the expansionduct from the subsidiary duct, the alginate droplets gel (optionallyentrapping or immobilizing, for instance, cells or genes suspendedwithin the alginate matrix) as calcium ions preferentially replace thesodium ions in the alginate. This example method produces gelled(non-exclusively) sphere-like droplets of highly precise and repeatabledimensions, not easily attainable by other means and lends considerableadvantage to the use of alginate which is widely used in food,pharmaceutical, textile, and paper products, exploiting the propertiesfor thickening, stabilizing, gel-forming, and film-forming. Forinstance, in gene delivery systems it is desirable to construct micronand nanometre sized capsules with high precision of morphologicaldimensions, thus assisting their targeted delivery to specific tissueswithin the mammalian body. As a further specific illustration,microparticles formed in this way, which are advantageously suitable forgene delivery, may be comprised of biocompatible bio-erodible hydrogelsincluding polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides,polyacrylic acid, alginate, chitosan, in which are suspended genes ofgreater than 30 nucleotides with specific expression promoters.

In accordance with another aspect, the fluid manipulation device may beused to function for the elution of liquid solutions and suspensionsinto gelled, polymerised, solidified materials in spherical volumesranging from 0.1 cubic millimetre to 0.003 cubic micrometers ofrepeatably high precision volume.

In accordance with another aspect, the fluid manipulation device may beconfigured such that a first duct, along which is caused to move asegmented flow stream of immiscible fluids, is temporarily merged withanother duct carrying one liquid phase component similar in phase to oneof the liquid phase components located within the first duct. After thepoint of merger the flow streams remain in a common duct before thenexiting through two separate ducts formed by splitting the common ductin two. At the point where the two ducts merge, the flow streams meetbut remain substantially separated due to the laminar flow resultingfrom the low Reynolds number conditions. Despite this laminar flow,there is an attraction between like fluids in the two streams that mergeat the junction, causing like fluids to coalesce and move out throughone exit duct and the unlike fluids to move out via the other exit duct.

In accordance with another aspect, the fluid manipulation device may beused for the creation of quantum dots, and assemblages thereof, from aliquid phase. To illustrate this process, an illustrative example isdescribed. In this embodiment, an organometallic precursortriocytlphosphine oxide (TOPO) are provided in separate immisciblefluids, formed on the device into segmented flow streams. The fluidsegments containing the triocytlphosphine oxide (TOPO) may beselectively heated by incorporating an energy absorbing dye (e.g. aninfrared-absorbing dye) or particulate material (titanium, platinum,palladium, to absorb light or induced electric fields) within the samefluid segments or the entire train of fluid segments may be heated bythe direct application of a heating element (e.g. electrical resistanceheater). Contiguous fluid segments in the segmented flow of the deviceenjoy a physical contact at the end of each fluid segment. As thesegments are caused to move along the reactor this interfacial boundaryis highly dynamic, and continually renewing. Mass transfer across thisinterfacial boundary can therefore be very rapid and is dependent uponseveral factors including the speed at which the segments are caused tomove in the device. The rate at which the organic precursor istransferred to adjacent fluid segments containing the triocytlphosphineoxide (TOPO) may be controlled by adjusting the flow rate of thesegmented flow stream. Equally, the mass transfer may be controlled byaltering the geometry of the duct carrying the segmented flow such thatan expansion (which may be for only a selected part of the duct) in thesize of the duct will cause elongate fluid segments to assume aspherical form as freely moving droplets that have no contact with theduct walls, and thereby do not enjoy the rapid internal vortex flow.Accordingly, mass-transfer is adjusted.

It will be apparent to those skilled in the art, given the benefit ofthis disclosure, that duct geometries may be designed to provideprecision control of mass-transfer and by which means assume accuratecontrol of quantum dot nucleation conditions. Quantum dots arenano-scale crystalline structures which can transform the colour oflight and have numerous applications ranging from computing, photonicand materials coating to biotechnology. The energy states of the dotscan be largely controlled by the size of the dot. Typically, this isachieved by introducing a desired organometallic precursor into heatedtriocytlphosphine oxide (TOPO) that has been vigorously stirred under aninert atmosphere. As nucleation proceeds to further shell growth thesolution changes through different colour regimes as the dot sizeincreases. Precision control of quantum dot formation has been difficultto refine because it is not readily possible to achieve ‘snap’ controlof nucleation thermal and other conditions. Subsequently, quantum dotsare usually sized post-synthesis to obtain particles of specificcharacter. Quantum dot formation in the device described offers astep-change advantage where thermal control may be exercised withconsiderable precision. In particular, with segmented flow the masstransfer can be almost instantaneously directed by switching betweenvortex flow and laminar flow, the latter in an expansion zone of theduct(s). In addition the fluoropolymer construction of the device allowsthe reaction to be conducted at sufficiently high temperatures, whichwould not be possible were it constructed from other polymers (e.g.polycarbonate), and the enclosed nature of the ducts can be utilised tocontrol the required inert atmosphere conditions. The processing can befurther elaborated by the introduction of further reagents throughtributary ducts to the main duct carrying the segmented flow stream, forinstance to alter the composition of subsequent layers around the coreof the quantum dot.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described by way ofexamples only and with reference to the accompanying drawings, in which:

FIGS. 1A-1E show several variants of a first embodiment of fluidmanipulation device in accordance with the invention;

FIGS. 2A-2B are sectional and plan views of a second embodiment of fluidmanipulation device;

FIGS. 3A-3B are plan and sectional views of a third embodiment of fluidmanipulation device;

FIGS. 4A-4B are plan and sectional views of a fourth embodiment of fluidmanipulation device;

FIGS. 5A-5D are views of a fifth embodiment of fluid manipulationdevice;

FIG. 6 is a side view of a daughterboard for fluid manipulation devicesof the present invention;

FIG. 7 is a plan view of a sixth embodiment of fluid manipulationdevice;

FIGS. 8A-8C show a seventh embodiment of fluid manipulation device;

FIGS. 9A-9C show variants of the device of FIGS. 8A-8C;

FIGS. 10A-10B show an eighth embodiment of fluid manipulation device;

FIGS. 11A-11B show variants of a ninth embodiment of fluid manipulationdevice;

FIG. 12 shows a tenth embodiment of fluid manipulation device;

FIG. 13 shows an eleventh embodiment of fluid manipulation device;

FIG. 14 shows a twelfth embodiment of fluid manipulation device;

FIGS. 15A-15C show a thirteenth embodiment of fluid manipulation device;

FIGS. 16A-16B show a fourteenth embodiment of fluid manipulation device;

FIG. 17 shows a fifteenth embodiment of fluid manipulation device;

FIG. 18 shows a sixteenth embodiment of fluid manipulation device;

FIG. 19 shows a seventeenth embodiment of fluid manipulation device; and

FIGS. 20A-20D show variants of an eighteenth embodiment of fluidmanipulation device.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIGS. 1A-1E show various configurations for a fluid manipulation device.In FIGS. 1A-1B a first fluoropolymer-based or -coated substrate 1 and asecond fluoropolymer-based or -coated substrate 2 are held in intimatejuxtaposition by various means. In FIGS. 1A-1E the substrates areencased within two or more additional layers of casement material 3. Thesubstrate layers sit within a cavity machined within the casementlayers, the depth of which is slightly shorter than the combinedthickness of the substrate layers. The casement material may extend, asprojections 4, through vias in the substrate layers joining compatibleprotrusions on another corresponding casement layer. The casementmaterial is a thermoplastic polymer welded together by thermocompressionbonding or by inductive heating, of an optional thin metallic film 5that may be applied to one of the two layers, to >Tg+5° C. of thethermoplastic polymer. At the points where the casement materials meet,the surface may be provided with interlocking inter-digitations usefulfor alignment, allowing for variations in the thickness of substratelayers and enhanced surface area for joining. Alternatively, thecasement layers 3 may be constructed from a rigid material such asstainless steel and bolted or clamped together. Incorporated within atleast one or both of the substrate layers there are at least two ducts6. These ducts are separated by a thin partition 90. The same ducts joina third duct the cross-sectional area of which is equal to the summedcross-sectional area of the two ducts 6. At a minimum distance 91 fromthe juncture of ducts 6 and 7 third duct 7 joins, axially, a fourth,segmented flow duct 10 that is of cross-sectional area and width atleast 25% that of the third duct 10, and most preferably 50% of that ofthe third duct. The geometrical restriction 9 in the common duct 7reduces the cross-sectional dimensions so that the laminar flow streamswithin the common duct convert to segmented flow 8 within the adjoiningsegmented flow duct 10. The fluid manipulation device or assemblyincorporates one or more radio frequency transponders 11 embedded withinits matrix or attached to the external surface. In FIGS. 1B-1E one ormore openings 12 within the substrate layers may be provided andconnected to the external environment with input and/or output ducts 13for the transfer of fluids. In FIGS. 1C-1D a second fluoropolymer-basedor -coated substrate is omitted and instead a fluoropolymer coating 14is applied to the layer of casement material that lies in intimatejuxtaposition with the first substrate layer. FIG. 1D shows a fluidmanipulation device as in FIG. 1C but where a thin metallic film 15 isdeposited on to the non-machined parts of the substrate layer andcasement layer. This film may be caused to rise in temperature byinductive heating and weld the substrate layer to the casement materialthat lies in intimate juxtaposition with it. FIG. 1E shows a cut-awayplan view of the fluid manipulation device showing features common toall variants of the fluid manipulation device, with the exception ofoptional input-output fluid communication ducts 13.

FIGS. 2A-2B show a fluid manipulation device similar to FIG. 1 but whereno separate casement material is provided. A first fluoropolymer-basedor -coated substrate 1 and a second fluoropolymer-based or -coatedsubstrate 2 are held in intimate juxtaposition by means ofthermocompressive welding or inductive heating of an optional metallicfilm 16 deposited on the non-duct surfaces, of at least one of thesubstrate layers, to at least 5° C. above the glass transitiontemperature of the fluoropolymer film or bulk material and subsequentlycooled to ambient.

FIG. 3 shows a preferred embodiment of the device for the manipulationof fluids. Casement layers 3 are provided with peripheralinter-digitated, interlocking, concentric semi-circumferential fins 17that enable the casement parts to join in a specific geometricalconfiguration, and allow for an enhanced mechanical binding betweenmating layers. Casement protrusions 4 reach through vias in thesubstrate layers and join corresponding protrusions from the opposedcasement layer. The casement layers may be provided with one or morethrough-holes 18 which allow additional functionalities. Thethrough-holes may provide visual access to the fluidic ducts insubstrate layers 1 or 2 where the nearest substrate layer is configuredto allow optical access, for example for a charged coupled device. Thethrough-holes are provided with an annular recessed undercut 19, whichcan be used to retain additional substrate devices fitted with acorresponding peripheral rim. The device may be provided with one ormore additional supporting layers 20 situated either side of thejuxtaposed substrate layers 1, 2.

FIGS. 4A-4B show another preferred embodiment of the device for themanipulation of fluids. More than one substrate layers 1 are stacked ontop of each other in an assembly. Substrate layers may have ductsmachined in one side or both. Fluidic communication between layers maybe provided by individual through-layer via holes translocating fluidsfrom one layer to the next or any other. Equally, fluid communication totwo or more layers may share a common opening for connection with theexternal environment which communicates to each layer through openingsin each layer that are in turn aligned vertically through the stack ofsubstrate layers as indicated in FIG. 4B. Such a vertical common duct 22may act as an output drain for fluids emerging from one or moresegmented flow ducts (not shown in FIG. 4 for clarity, but see 10 inFIG. 1) on two or more substrate layers. The assembly may accommodate avariable number of substrate layers by incorporating a casement layerspacer 23 which may be thermally joined or bolted to casement layers 3.

FIGS. 5A-5D show another preferred embodiment of the device for themanipulation of fluids. The Figure shows a device similar to thatdescribed in FIG. 4 but where the through-holes 18 in casement 3incorporate one or more smaller substrate devices (called hereafterdaughterboards 24 as described in more detail in FIG. 6) or multilayerdaughterboards 35 of such. FIG. 5B shows side and plan views of adaughterboard showing the distal surface 36 with protrusions 27 and theproximal surface 37 with through vias 28. FIG. 5C shows a single layerdaughterboard 24 and a multilayer daughterboard 35. The multilayerdaughterboard may be formed from a daughterboard substrate withdaughterboard interlayers 38 stacked on top of one another. Thedaughterboard interlayers may be provided with a range of through viasaligned with the substrate layer protrusion vias 28 for the passage offluids between layers or additional through vias for further fluidcommunication between layers or alternate sides of given interlayers. Afurther daughterboard capping layer 39 may be provided which contains nothrough vias but may incorporate ducts for the passage of fluids. FIG.5D shows a fluid handling device as described more fully in FIG. 4 butwhere the casement through holes have been occupied with an exampledaughterboard 24 and a daughterboard substrate assembly 35.

FIG. 6 shows a preferred embodiment of the daughterboard. Eachdaughterboard comprises a substrate base 25 fitted with a recessed rim26 so that it can be secured in place by the recessed undercut 19 incasement 3 through-holes 18 (see FIG. 3 for 3, 18, 19). A daughterboardmay comprise one or more protrusions 27 from the base, in each of whichprotrusions a via 28 may be provided. The daughterboard substrate viasmay function for the passage of fluids, or with appropriate coatings theguided passage of light through a liquid core within the via, or withappropriate metallic or conductive polymer coatings the communication ofelectrical power or data. The daughterboard may have machined within itsproximal surface one or more ducts 29 for the passage of fluids. Thedaughterboard substrate may be bonded to another daughterboard cappinglayer 30 (called herein daughterboard capping layer) in which furtherducts 31 may be provided for the passage of fluids. The daughterboardsubstrate layer may be provided with cavities 32 accessible from thedistal side in which can be placed sensor and actuator components. Thedaughterboard substrate layer may be provided with an optional alignmentridge 33 that can mate with a corresponding groove (not shown forclarity) in the daughterboard capping layer. The daughterboardprotrusions 27 may be provided with compressible fluoroelastomericsealing rings 34 to help provide hermetic seals when interfaced with thefluid manipulation device.

FIG. 7 shows another preferred embodiment of a fluid manipulationdevice. In this embodiment, fluid inputs are made from centralised fluidcommunication ports and segmented fluid flow through the network ofducts is caused by centrifugal means. Immiscible fluids are caused tomove from an external source through fluid communication ducts 13 to andthrough the ducts 6 to a common duct 7 containing a geometricalrestriction 9, or other device to cause segmentation of laminar fluidstreams, and into the segmented flow duct 10. The segmented flow streamin duct 10 may be further divided into two or more segmented flow ductswhich exit the fluid manipulation device directly from the substratelayer 3 through a peripheral exit port 40 located at the periphery ofthe device or through a duct 13 in casement 3 on either the proximal ordistal surface. Two or more segmented flow ducts may merge into onecommon duct draining segmented flow streams from the device.

FIG. 8A shows two ducts 6 along which immiscible fluids are caused tomove into a common duct 7 which incorporate a geometrical constriction 9that converts laminar flow in common duct 7 to segmented flow 8 insegmented flow duct 10. The relative volumes of the immiscible phaseliquid components present in the segmented flow stream may be controlledby the relative flow rates of the immiscible phase liquids. FIGS. 8B-8Cshow variants of FIG. 8A but where the relative proportions of theimmiscible phase liquids in the segmented flow duct have been altered bythe flow rates of the same liquid phase components through the inputducts 6. This enables liquids to, be eluted in the segmented flowduct(s) as serial aliquots of defined volume.

FIG. 9A shows two ducts 6 along which immiscible fluids are caused tomove into a merged common duct 7 which incorporates a segment 41provided with a change in surface energy that converts laminar flow incommon duct 7 to segmented flow 8 in segmented flow duct 10. FIG. 9Bshows two ducts 6 along which immiscible fluids are caused to move intoa merged common duct 7 which incorporates a segment provided with aphoto-responsive surface 42 in contact with the fluid and of whichsurface the contact angle varies upon illumination 43 so causing thelaminar flow in common duct 7 to convert to segmented flow 8 insegmented flow duct 10. The effect of illumination is to cause atemporally defined and reversible photo-wetting effect. FIG. 9C showstwo ducts 6 along which immiscible fluids are caused to move into amerged common duct 7 which incorporates a segment provided with twoelectrically conductive surfaces 44 separated by an insulator, whichcould be the wall of the duct or a thin interelectrode film, with one ofthe surfaces in contact with the liquids in common duct 7. Uponelectrical polarisation with an applied voltage 45, the contact anglevaries causing the laminar flow in common duct 7 to convert to segmentedflow 8 in segmented flow duct 10. The effect of polarisation is to causea temporally defined and reversible electro-wetting effect.

FIGS. 10A-10B show two ducts 6 along which immiscible fluids are causedto move into a merged common duct 7 which incorporates a cruciformjunction 46 or staggered-T or staggered-side (shown in FIG. 10B)junction with other ducts 47 that function to provide electro-kineticdisruptions of the parallel fluid flow in common duct 7 causing theconversion to segmented flow 8 in duct 10. Electrical potentials areapplied between the ducts 47 to cause electrokinetic flow across duct 7.

FIGS. 11A-11B show examples of a preferred fluid duct configuration forthe fluid manipulation device where in FIG. 11A two ducts 6 merge toform a common duct 7, and where in FIG. 11B three ducts 6 merge to forma common duct 7. In both examples laminar flow predominates withincommon duct 7 until the point of geometrical constriction 9 is reachedwhich causes laminar flow to convert to segmented flow 8 in segmentedflow duct 10. It will be realised by those practised in the art, giventhe benefit of this disclosure, that more than three ducts may also bemerged to form a common duct, so providing a means to produce streams ofsegmented fluids where even more than three immiscible phase componentsare present. In addition, a step-change in surface energy, photowetting,electrowetting and electrokinetic disruption of laminar immiscible phaseflow (as shown in FIGS. 9A, 9B, 9C, 10A and 10B) may be employed to formsegmented flow instead of constriction 9.

FIG. 12 shows a further example of another fluid duct configuration ofthe device for the manipulation of fluids as shown and described inFIGS. 8A-8C, but with the addition of another geometrical constriction48 within the segmented flow duct 10. This has the effect to cause achange in two- and three-dimensional morphology of the fluid segments 8as they pass through the constriction. This may convert, for example,ovoid fluid segments (as suggested in 8) to longer and thinner segments49. The constriction 48 may be repeated in a serial manner, soincrementally altering the morphology of fluid segments.

FIG. 13 shows a further example of another fluid duct configuration ofthe device for the manipulation of fluids. Here segmented flow stream 8is generated as in FIG. 1E but, equally, the same may be generatedaccording to the duct configurations shown in FIGS. 9-11. The segmentedflow duct splits at junction 50 into two or more smaller ducts 51,narrower in width, depth, both or diameter, causing the fluid segmentsto divide into smaller segments in each of the provided ducts 51.Optionally, the segmented flow duct 10 may be widened, as shown at 52,prior to splitting into the two or more smaller ducts, allowing forfluid segments to assume a morphology more conducive to splitting at thejunction 50.

FIG. 14 shows a further example of another fluid duct configuration ofthe device for the manipulation of fluids. Here segmented flow stream 8is generated as in FIG. 1E but, equally, the same may be generatedaccording to the duct configurations shown in FIGS. 9-13. The segmentedflow duct 10 is joined by one or more tributary ducts 53 at right anglesor any other angle allowing for additional liquids to pass into thesegmented flow duct. This feature facilitates the sequential exposure offluid segments to further liquids as they are caused to move along thesegmented flow duct.

FIG. 15 shows a further example of another fluid duct configuration ofthe device for the manipulation of fluids. In FIG. 15A fluid segments 8are caused to flow in segmented flow duct 10. Segmented fluids may thenbe caused to flow into an expansion duct 54 where the internaldimensions are expanded such that fluid segments, previously of anon-spherical morphology (as indicated in 8), assume a sphericalgeometry 55 where the periphery of the spherical segments no longermakes physical contact with the internal walls of the duct. By thismeans sequences of spherical droplets are formed.

It will be understood by those practiced in the art, given the benefitof this disclosure, that sequences of near-identical spheres can becreated, or alternatively sequences of custom size spheres, by alteringthe relative flow rates of the immiscible phase fluids caused to flowinto the common duct. FIG. 15B shows a similar example where the bulkmaterial or outer layer(s) of the spheres thus created may becross-linked or polymerised by exposure to electromagnetic radiation 56,such as ultraviolet light. FIG. 15C shows another similar example wherethe bulk material or outer layer(s) of the spheres thus created may becaused to solidify, gel, freeze, polymerise, crosslink or otherwise toassume a less than completely liquid state by exposure to additionalreagents fed from a tributary duct 57. Equally, the spheres may remainin a liquid form but become exposed to or enveloped by other compoundsprovided by the liquid added through the tributary duct. It will berecognised by those skilled in the art, given the benefit of thisdisclosure, that more than one tributary duct may be provided in aserial succession thus enabling the opportunity to make sequentialexposures to more than one additional reagent as may be required in aparticular chemical reaction, or required for the provision of layeredcoatings around each sphere. Linear arrows denote direction of fluidmovement. Waved arrows denote direction of exposure of electromagneticradiation.

FIG. 16 shows a further example of another fluid duct configuration ofthe device for the manipulation of fluids. The segmented flow duct 10may be provided with a tributary duct 61 that makes parallel contactover a short geometrical section 60 such that the fluids in both ductsflow in parallel in a shared duct for a short duration. In FIG. 16A acontactor duct contains an aqueous liquid phase 59 (shaded black). Atthe merger of the two ducts the aqueous component 59 of the segmentedfluid in the segmented fluid duct 10 is attracted to the aqueous fluid59 in the contactor duct and flows out through the continuation 62 ofcontactor duct with the rest of the aqueous fluid. The segmented fluidis thereby reduced to a singular stream of the other non-aqueous phase.In FIG. 16B the reverse is demonstrated whereby at the merger of the twoducts the non-aqueous component 58 of the segmented fluid in thesegmented fluid duct is attracted to the non-aqueous fluid 58 in thecontactor duct 61 and flows out with the rest of the non-aqueous fluidthrough continuation 62 of the contactor duct. The segmented fluid isthereby reduced to a singular stream of the aqueous phase. Theconfiguration of fluid ducts provides a means of reverting the segmentedflow stream back to the original separate phase components.

FIG. 17 shows a further example of another fluid duct configuration ofthe device for the manipulation of fluids. The gas-liquid phasesegmented flow in duct 10 may be interfaced with another duct 66 butseparated at the juncture by a gas permeable material 63 such as amembrane. The gas phase component 65 (shaded black) of the segmentedflow stream in duct 10 rapidly diffuses across membrane 63 and escapesthrough duct 65. This causes the segmented flow in duct 10 to revert toa continuous stream of liquid phase component in continuation of fluidduct 10 beyond the interface with duct 66.

FIG. 18 shows a further example of another fluid duct configuration ofthe device for the manipulation of fluids. The device shown isconfigured such that two separate aqueous fluid streams 67, 68 arecaused to segment with an immiscible fluid phase 72 at a constrictionjunction within separate common ducts 7. The two separate segmented flowstreams 8 are caused to move along separate segmented flow ducts 10 andwhich meet at junction 69. The segmented flow streams are appropriatelysynchronised by means of pumping systems applied to cause the aqueoussegments to meet and merge at the junction 69, to form larger aqueoussegments 70 separated by the immiscible phase components which alsomerge to form larger segments 71. The rapid internal circulation vortexflow within the fluid segments 70 and 71 cause very rapid mixing. Theaqueous segments or other immiscible phase components may comprisesimilar solutions incorporating dissolved or suspendedcompounds/components of similar or dissimilar concentrations. By themeans of controlled volume elution and rapid micromixing achieved withthe device configuration, a wide range of the dilution and/or mixingprotocol may be achieved. The Figure shown illustrates the device wheretwo segmented flow streams are merged, but others with more than 2segmented flow streams may be constructed.

FIG. 19 shows a further example of another fluid duct configuration ofthe device for the manipulation of fluids. Ducts 6 provide immisciblefluids which merge as parallel flow in common duct 7 before formingsegmented flow 8 in segmented flow duct 10 after constriction 9. One ofthe ducts 6 is joined by another duct 73 which feeds an additionalstream of the same liquid phase component. In the example shown, twofluid streams 74 and 75 of the same phase merge and form parallel flowin the merged duct 76. The output from the merged duct 76 in turn mergeswith two more ducts 6 containing immiscible phase 72 to form a commonduct 7. The constriction 9 in the common duct 7 causes the laminar flowof fluids 74, 75 and 72 to reform as segmented flow 8 in segmented flowduct 10. This geometrical configuration provides a means for theprecision controlled elution and mixing of similar or dissimilar fluidstreams, and is readily controllable over a wide range of flow rateconditions.

FIGS. 20A-20D show a further example of another fluid duct configurationof the device for the manipulation of fluids. The device is providedwith one or more pressure sensors in the form of capillary gas-filledmanometers. These are constructed as blind tributary ducts 80 arrangedat any angle between 0° and 180° to the first microfluidic duct 81 alongwhich fluids are caused to move. The first microfluidic duct may be anyduct formed on the device for manipulation of fluids where there isnon-segmented flow. The tributary ducts are preferably filled with inertgas such as argon or helium. Fluid within the first fluidic duct willrise into the tributary duct according to the prevailing pressureconditions. The tributary ducts function according to the principle thata change in gas volume provides a change in fluid pressure, i.e. anincrease in fluidic pressure gives a decrease in gas volume. Therefore,by knowing the cross sectional area and length of the blind tributaryducts, the initial volume can be calculated and with the displacement ofthe fluid in the channel, the increase/decrease of the pressure-affectedvolume can be calculated. With liquid in the first microfluidic duct 80,the liquid level within the tributary duct 80 rises to an initialpressure setting Pi. According to pressure changes (for example, duringfluid movement caused by the application of pressure) the pressure ofthe fluid increases and the fluid moves further up the tributary ductchanging the volume of the trapped gas, thus giving P1, P2, P3 pressurereadings (FIG. 20B). Other geometrical arrangements of more than onetributary duct 80 can provide information on fluid flow rate byindicating the pressure drop over predetermined length (FIG. 20C) orover a fluid resistor caused by a constriction 82 in the first fluidduct (FIG. 20D). The capillary manometers may be provided in a linear,serpentined, curved or coiled format and meniscus movements visualisedby an optical device such as a charged coupled device. Arrows denotedirection of fluid flow.

In any of the segment-generating devices in accordance with theinvention, the inlet or supply passages for the respective fluids may bedevoid of functionally operative input ports and so act as closedreservoirs (although these passages may have input ports which are usedduring manufacture and then sealed). Thus, the fluids are stored untilcaused to flow along their respective passages and combine to form asegmented flow: the segmented flow may pass out of the device through anoutput port, either to waste or to another device, or may pass to areservoir in the same device.

1-20. (canceled)
 21. A device comprising first and second inlet passagesfor respective immiscible fluids, the first and second inlet passagesmerging into a third passage along which, in use, the two fluids flowunder parallel laminar flow conditions, the third passage being formedwith a constriction or other discontinuity, in use, causing the twofluids to form into a flow of alternate segments.
 22. A device asclaimed in claim 21, in which downstream portions of the inlet passagesextend parallel with each other before merging to form the thirdpassage.
 23. A device as claimed in claim 21, in which said otherdiscontinuity in the third passage comprises a region of changed surfaceenergy.
 24. A device as claimed in claim 21, in which said otherdiscontinuity comprises a region of altered or alterable contact angle.25. A device as claimed in claim 21, in which said other discontinuitycomprises one or more further passages joining the third passage.
 26. Adevice as claimed in claim 21, comprising a further inlet passage for athird fluid, the further inlet passage merging into the third passageupstream of the constriction or other discontinuity.
 27. A device asclaimed in claim 21, in which the third or outlet passage is formed witha second constriction or other discontinuity downstream of the firstconstriction or other discontinuity.
 28. A device as claimed in claim21, wherein the surfaces of the third passage which, in use, are incontact with said fluids comprise a fluoropolymer.
 29. A device asclaimed in claim 21, further comprising a source of electromagneticradiation for polymerising or cross-linking the content (or part) ofliquid segments produced downstream of the constriction or otherdiscontinuity.
 30. A device as claimed in claim 21 wherein the thirdpassage is provided with an enlargement in cross-section downstream ofthe constriction or other discontinuity.
 31. A method of producing asegmented flow of first and second immiscible fluids comprising (i)providing a device with a first conduit provided with a constriction orother discontinuity (ii) causing the first and second immiscible fluidsto flow under laminar flow conditions along said first conduit, whereinthe constriction or other discontinuity causes the first and secondimmiscible fluids to form into a flow of alternate segments downstreamof the constriction or other discontinuity.
 32. A method as claimed inclaim 31 wherein the device is provided with first and second inletpassages for the delivery of the first and second immiscible fluidsrespectively to the first conduit.
 33. A method as claimed in claim 31wherein the first and second inlet passages merge into the firstconduit.
 34. A method as claimed in claim 31 wherein the first andsecond inlet passages merge into the first conduit and downstreamportions of the first and second inlet passages extend parallel witheach other before merging to form the first conduit.
 35. A methodaccording to claim 31 wherein the surface of said first conduit incontact with said first and second immiscible fluids comprises afluoropolymer.
 36. A method according to claim 31 wherein the flow ratesof the first and second immiscible fluids in the first conduit aremutually different.
 37. A method according to claim 31 wherein the firstand second immiscible fluids are exposed to ultra-violet radiationdownstream of the constriction or other discontinuity.
 38. A methodaccording to claim 31 wherein the contents (or part) of liquid segmentsare polymerised or cross-linked by exposure to electromagneticradiation.
 39. A method as claimed in claim 31 comprising causingsegments of at least one of the first and second fluids to form asubstantially spherical shape.
 40. A fluid manipulation devicecomprising first and second ducts for the passage of respectiveimmiscible fluids, each of said first and second ducts having arespective inlet for the introduction of said respective fluid into saiddevice, wherein said first and second ducts join to form a third ductalong which, in use, the first and second fluids flow under laminar flowconditions, the third duct being formed with a constriction, theconstriction causing, in use, the first and second fluids to form into aflow of alternate segments, wherein the device comprises two substratesdisposed face-to-face, the surface of at least one of the substratesbeing profiled such that the first, second and third ducts are definedbetween the two substrates, the surfaces of the third duct that, in use,comes into contact with one or both of the first or second fluidcomprising a fluoropolymer.
 41. A fluid manipulation device according toclaim 40 wherein the surfaces of the first and second ducts that, inuse, are in contact with the respective first and second fluids comprisea fluoropolymer.
 42. A fluid manipulation device according to claim 40wherein the two substrates are held together by outer members.